1. Field of the Invention
The present invention relates generally to the study of Wnt signaling and lytic bone disease. More specifically, the present invention discloses methods for repairing lytic lesions and inhibiting tumor progression using Wnt ligands with or without anti-DKK1 therapy.
2. Description of the Related Art
Wnts comprise a highly conserved family of secreted glycoproteins consisting of 19 members. Wnt ligands bind to the Frizzled receptors alone or complexed with the low-density lipoprotein receptor-related proteins (LRP) 5/6. In vertebrates, Wnts can activate a ‘canonical’ b-catenin-dependent pathway or several b-catenin-independent ‘noncanonical’ pathways (Nusse, 2005; Veeman et al., 2003). The canonical Wnt-b-catenin pathway is normally repressed at several levels. An intracellular complex including GSK-3, axin, and the tumor suppressor gene product APC function to phosphorylate b-catenin, which in turn targets it for ubiquitin-mediated proteasomal degradation. Upon Wnt binding, b-catenin degradation is blocked leading to its accumulation and translocation to the nucleus, where it binds the TCF/LEF family of transcription repressors turning them into transcriptional activators (Nusse, 2005; van de Wetering et al., 2002).
Mounting evidence suggests that canonical Wnt signaling is central to normal skeletogenesis (Day et al., 2005; Kirstetter et al., 2006) and cancer related bone diseases (Johnson and Rajamannan, 2006; Stewart and Shaughnessy, 2006). The first direct evidence of role of Wnt signaling in bone formation came from seminal observations that inactivating mutations of the LRP5 gene caused osteoporosis-pseudoglioma syndrome (OPPG) (Gong et al., 2001). Subsequently, it was shown that a separate and distinct mutation in the same gene results in high bone density (Boyden et al., 2002; Little et al., 2002). Expression of Wnt10b in transgenic mice increased bone mass (Bennett et al., 2005) and overexpression of Wnt7B and beta-catenin in C3H10T1/2 osteoblastic precursor cells induced their differentiation into mature osteoblasts (Hu et al., 2005; Rawadi et al., 2003). Osteoclastogenesis is primarily regulated by receptor activator of the NF-kB ligand (RANKL) binding to RANK on the surface of osteoclast precursor cells. The ability of RANKL to bind RANK, and hence promote osteoclast development is tightly regulated by the RANKL decoy receptor, osteoprotegerin (OPG) (Lacey et al., 1998; Simonet et al., 1997). Remarkably, recent studies have shown that Wnt signaling in cells of the osteoblast lineage positively regulates the expression of OPG (Glass et al., 2005; Holmen et al., 2005) while negatively regulating RANKL (Spencer et al., 2006). Taken together these studies suggest that Wnt signaling is likely to be a central regulator of bone remodeling through its direct effects on osteoblastogenesis and indirect effects on osteoclastogenesis.
Multiple myeloma (MM) is a malignancy of plasma cells that uniquely accumulate in the bone marrow (BM), but not other organ systems, suggesting that the bone marrow provides unique growth and survival signals for MM cells. MM is characterized by osteolytic bone disease, which is caused by an uncoupling of bone remodeling as a result of increased osteoclast activity and decreased osteoblast activity (Bataille et al., 1991; Roodman, 2004; Taube et al., 1992). Over the last three decades numerous experimental and clinical studies have focused on the role of osteoclasts, and the identification of critical factors associated with increased osteoclast activity in MM (Sezer et al., 2003). However, it was recently observed that suppression of osteoblastogenesis by the soluble Wnt signaling inhibitor, Dickkopf-1 (DKK1), likely played critical role in MM-induced bone disease (Tian et al., 2003). DKK1 is the prototypical member of a family of secreted glycoproteins capable of inhibiting canonical Wnt signaling by binding to LRP5/6 causing it to be internalized and degraded (Binnerts et al., 2007). Wnt ligand interaction with its receptor can also be regulated by the secreted frizzled related proteins (sFRPs). As their name suggests, these factors are decoy receptors with frizzled domains capable of binding Wnts in solution (Finch et al., 1997). Interestingly, MM cells produce sFRP-2 (Oshima et al., 2005) and FRZB/sFRP-3 (de Vos et al., 2001; Zhan et al., 2002) and these factors may also contribute to the suppression of Wnt signaling in the bone marrow microenvironment (Stewart and Shaughnessy, 2006). It was recently shown that administration of a neutralizing antibody to DKK1 to myelomatous bones, presumably resulting in enhancement of Wnt signaling in the bone, prevented bone resorption and also tumor progression (Yaccoby et al., 2006). The importance of DKK1 secretion in diseases associated with bone destruction is reinforced by recent studies showing that a neutralizing antibody to DKK1 could inhibit the bone destructive process in rheumatoid arthritis (Sen, 2005).
As Wnt signaling activation has been linked to several forms of cancer, including blood malignancies (Polakis, 2000), the observation that neutralizing DKK1, and therefore enhancing Wnt signaling, has negative effects on MM tumor growth in bone, might be considered counterintuitive. However, it is now well recognized that an interaction between tumors and their associated stroma can dramatically influence tumor behavior (Karnoub et al., 2007). Therefore, it is possible that activation of the Wnt signaling pathway in the tumor-stroma milieu might override the effects of activating that signaling pathway in tumor cells when they are grown in a relatively static tissue culture environment.
Various clinical observations (Coleman et al., 2005) and experimental studies (Pearse et al., 2001; Vanderkerken et al., 2003; Yaccoby et al., 2004) have linked the level of MM bone disease with tumor progression. The notion that bone disease drives MM progression, at least in the initial phases of the disease process is also supported by studies demonstrating that osteoclasts alone can support long term survival and proliferation of primary MM cells (Yaccoby et al., 2004; Abe et al., 2004) and that osteoblastic cultures impede growth of MM cells from a large subset of patients (Yaccoby et al., 2006).
Despite this, the proliferative effects of Wnts in multiple myeloma are controversial. For instance, one study showed that Wnt could promote proliferation of multiple myeloma cells (Derksen et al., 2004) while another study was unable to show this effect (Qiang et al., 2003; Qiang et al., 2005). Thus, prior art lacks understanding of the role played by Wnt signaling in multiple myeloma and multiple myeloma-induced lytic bone disease. Additionally, prior art is deficient in means of repairing lytic lesions in patients with multiple myeloma and other cancers (breast cancer, prostate cancer) and inhibiting progression of the tumor. The present invention fulfills this longstanding need and desire in the art.